Method of treating surface of substrate used in biological reaction system

ABSTRACT

Provided is a method of treating a surface of a substrate used in a biochemical reaction system, the method including forming a polymer film on the surface by vapor deposition of a compound of formula (1) below and a compound of formula (2) below: 
       (RO) 3 —Si—(CH 2 ) n1 —X  (1) 
       (RO) 3 —Si—(CH 2 ) n2 —(CF 2 ) m —X  (2) 
     wherein R is one of a methyl group and an ethyl group, X is one of a methyl group and a trifluoromethyl group, n1 is an integer from 1 to 3, n2 is an integer from 1 to 10, and m is an integer from 1 to 10.

This application is a divisional application of U.S. patent applicationSer. No. 10/756,366, filed Jan. 27, 2004, which claims priority to andthe benefit of Korean Patent Application No. 2003-5486, filed on Jan.28, 2003, in the Korean Intellectual Property Office, the disclosure ofwhich is incorporated herein in its entirety by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method of treating a surface of asubstrate used in a biological reaction system, and more particularly,to a method of chemically treating a surface of a substrate used in abiological reaction system to prevent biological molecules from adheringto the surface.

2. Description of the Related Art

Most miniaturized labs-on-a-chip consist of a DNA extraction or samplepreparation unit, a DNA amplification unit, and a DNA detection unit.The DNA amplification unit includes a thermal cycler that repeatsheating and cooling to a denaturation temperature, an annealingtemperature, and an extension temperature to amplify DNA.Conventionally, a polymerase chain reaction (PCR) system has been madeof a polypropylene e-tube. Recently, silicon or glass has been used moreand more for the PCR reactor. The surface area per volume of the PCRreactor increases more and more. As a result, non-specific binding ofPCR reactants and products to the surface of the PCR reactor occurs morefrequently, lowering the yield of the PCR reaction.

Silicon and glass can be utilized as materials for various biochemicalreactors, in addition to the PCR reactor. Accordingly, these biochemicalreactors also suffer from non-specific binding of biomolecules to thesilicon or glass surface and yields low from the biochemical reaction.Therefore, in biochemical reactors made of silicon or glass with a PCRreaction therein, it is required to treat a silicon or glass surface ofthe reactor to prevent non-specific binding of biomolecules.

Exemplary conventional suggestions for preventing a non-specific bindingof biomolecules to the surface of a silicon structure, including a PCRreactor, where biochemical reactions take place, include forming a SiO₂film on the surface of the silicon structure by oxidization at hightemperature and depositing a polymer solution that can suppress suchnon-specific binding to the surface of the silicon substrate.

U.S. Pat. No. 6,475,722 discloses the formation of a silicon oxide filmor a silicon nitride film on a silicon surface of a DNA processingsystem, which includes PCR reactor, to prevent a non-specific absorptionof DNA or other biochemical molecules to the silicon surface.

U.S. Pat. No. 6,261,431 discloses the addition of bovine serum albumin(BSA) into a PCR buffer. A method of adding a dispersant, such as Tween20, together with BSA to lower surface energy of the buffer was alsosuggested (Ivonne Schneegab et al., “Miniaturized flow-through PCR withdifferent types in a silicon chip thermocycler,” Lab-on-a-chip, Vol. 1,p. 42-49, 2001)

U.S. Pat. No. 6,156,389 discloses a method of processing a silicon orglass surface to be hydrophobic, in which the silicon or glass surfaceis coated with a solution of a fluorinated monomer that contains 3 to 20carbon atoms and a trifluoromethyl group at least one terminal byspraying or dipping. However, the patent aims only at making the surfacehydrophobic and, evidently from many experimental data, rather leads toa lower yield from the PCR reaction compared to before the surfacetreatment that results in only a kind of fluorinated hydrocarbon film(Nucleic Acids Research, “Chip PCR. 1. Surface Passivation ofMicrofabricated Silicon-glass Chips for PCR”, 24, 1996, 375-379).

In addition, there is a method of using a polymer solution which cansuppress such a non-specific adsorption. This method involves injectingthe polymer solution into a miniaturized 3D chip structure to coat achip surface and rinsing and drying processes. These processes areexperimentally complicate to perform and the results are notreproducible (Nucleic Acids Research, 24, 1996, “Chip PCR.1. SurfacePassivation of Microfabricated Silicon-glass Chips for PCR”, 375-379;Clinical Chemistry, 41, 1995, “Thermal Cycling and Surface Passivationof Micromachined Devices for PCR Chip”, 1367-1368).

SUMMARY OF THE INVENTION

The present invention provides a method of treating a surface of asubstrate used in a biochemical reaction system, such as a polymerasechain reaction (PCR) chip.

The present invention also provides a composition for treating a surfaceof a substrate used in a biochemical reaction system.

In one aspect of the present invention, there is provided a method oftreating a surface of a substrate used in a biochemical reaction system,the method comprising forming a polymer film on the surface by vapordeposition of a compound of formula (1) below and a compound of formula(2) below:

(RO)₃—Si—(CH₂)_(n1)—X  (1)

(RO)₃—Si—(CH₂)_(n2)—(CF₂)_(m)—X  (2)

wherein R is one of a methyl group and an ethyl group, X is one of amethyl group and a trifluoromethyl group, n1 is an integer from 1 to 3,n2 is an integer from 1 to 10, and m is an integer from 1 to 10.

The compound of said formula (1) and the compound of said formula (2)are simultaneously deposited by vaporization. Alternatively, thecompound of said formula (1) and the compound of said formula (2) may besequentially deposited by vaporization. The vapor deposition may becarried out at a temperature of 60-140° C.

Non-limiting materials for the substrate may include silicon, glass,etc. The substrate treated by the above method may be used inbiochemical reaction systems, such as PCR chips.

In another aspect of the present invention, there is provided acomposition for treating a surface of a substrate used in a biochemicalreaction system, the composition comprising a compound of formula (1)below and a compound of formula (2) below:

(RO)₃—Si—(CH₂)_(n1)—X  (1)

(RO)₃—Si—(CH₂)_(n2)—(CF₂)_(m)—X  (2)

wherein R is one of a methyl group and an ethyl group, X is one of amethyl group and a trifluoromethyl group, n1 is an integer from 1 to 3,n2 is an integer from 1 to 10, and m is an integer from 1 to 10.

The above composition may be applied to the treatment of a surface of asilicon or glass substrate that is utilized in a biochemical reactionsystem, especially a PCR chip.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other features and advantages of the present inventionwill become more apparent by describing in detail exemplary embodimentsthereof with reference to the attached drawings in which:

FIG. 1 illustrates the formation of a polymer film on a surface of asilicon substrate by a surface treatment method according to the presentinvention;

FIG. 2 is a graph of the yield of polymerase chain reaction (PCR) for asilicon chip that is surface-treated according to Example 1;

FIG. 3 is a graph of the yield of PCR for a silicon chip that issurface-treated according to Comparative Example 1;

FIG. 4 is a graph of the yield of PCR for a silicon chip that issurface-treated according to Comparative Example 2; and

FIG. 5 is a graph of the yield of PCR for a polypropylene e-tube used asa control.

DETAILED DESCRIPTION OF THE INVENTION

In a method of treating a surface of a substrate used for a biochemicalreaction system according to an embodiment of the present invention, apolymer film is formed on the substrate by simultaneous vapor depositionof a compound of formula (1) below and a compound of formula (2) below,both of which contain a silane group. Alternatively, the polymer filmmay be formed by sequential vapor deposition of the compound of formula(1) and the compound of formula (2) on the substrate.

(RO)₃—Si—(CH₂)_(n1)—X  (1)

(RO)₃—Si—(CH₂)_(n2)—(CF₂)_(m)—X  (2)

In formulae (1) and (2) above, R is a methyl group or an ethyl group, Xis a methyl group or a trifluoromethyl group, n1 is an integer from 1 to3, n2 is an integer from 1 to 10, and m is an integer from 1 to 10.

When the compound of formula (1) and the compound of formula (2) aresimultaneously coated on the surface by vapor deposition, a compositionfor the coating may include 40-60% by weight of the compound of formula(1) and 40-60% by weight of the compound of formula (2).

As the compound of formula (1) and the compound of formula (2) aresimultaneously or sequentially coated on the substrate, which is made ofsilicon or glass, by vapor deposition, a polymer film is formed by thepolymerization of the two compounds. Vapor deposition may be performedby vaporization at a low temperature of 60-140° C. The surface of thesubstrate may be activated by UV irradiation prior to the deposition ofthe compound of formula (1) and the compound of formula (2).

The substrate to be surface-treated for use in a biochemical reactionsystem may be a glass or silicon substrate. However, materials for thesubstrate are not limited to these materials.

As described above, the polymer film is formed by polymerization whilethe hydrocarbon compounds that commonly contain a silane group but havedifferent chain lengths are coated on the surface of the substrate byvapor deposition. The polymer film has a hydrophobic surface due to aterminal —CF₂ or CF₃ group of the hydrocarbon chains. This hydrophobicsurface has no affinity to biological materials, including DNA.

A process of forming the polymer film on a surface of a siliconsubstrate by the surface treatment method according to the presentinvention is illustrated in FIG. 1.

The present invention also provides a biochemical reaction systemmanufactured using a substrate that is surface-treated by the abovemethod. A representative example of the biochemical reaction system is apolymerase chain reaction (PCR) system. However, the biochemicalreaction system according to the present invention is not limited to thePCR system and may be any biochemical reaction system that treats ofbiochemical materials, such as nucleic acid, protein, etc.

A composition for treating a surface of a substrate used in abiochemical reaction system according to the present invention containsthe compound of formula (1) and the compound of formula (2). Thecomposition may contain 40-60% by weight of the compound of formula (1)and 40-60% by weight of the compound of formula (2).

The present invention will be described in greater detail with referenceto the following examples. The following examples are for illustrativepurposes and are not intended to limit the scope of the invention.

EXAMPLE 1

A silicon substrate with native oxide was pre-treated in an ozonereactor to remove impurities and form silanol groups on the surfacethereof.

A Teflon chamber was placed in an oven set at 85° C. at which compoundshaving silane groups could vaporize. A container containing a compoundhaving the formula of (MeO)₃—Si—(CH₂)—CH₃ (available from Aldrich) wasput into the Teflon chamber, and the compound was vaporized to saturatethe Teflon chamber.

The silicon substrate with the silanol groups was put into the Teflonchamber and subjected to vapor deposition for 10 minutes. The containerin the Teflon chamber was replaced with a container containing acompound having the formula of (MeO)₃—Si—(CH₂)₂—(CF₂)₇—CF₃ (availablefrom Shin-etsu Chemical.), and vapor deposition was further conductedfor 1 hour.

EXAMPLE 2

The surface of the silicon substrate was treated in the same manner asin Example 1, except that a 1:1 composition of (MeO)₃—Si—(CH₂)—CH₃ and(MeO)₃—Si—(CH₂)₂—(CF₂)₅—CF₃ was used.

COMPARATIVE EXAMPLE 1

A silicon substrate with native oxide or artificial thermal oxide waspre-treated in an ozone reactor to remove impurities and form silanolgroups on the surface thereof.

A Teflon chamber was placed in an oven set at 85° C. at which compoundshaving silane groups could vaporize. A container containing a compoundhaving the formula of (MeO)₃—Si—(CH₂)₂—(CF₂)₇—CF₃ (available fromShin-etsu Chemical.) was put into the Teflon chamber, and the compoundwas vaporized to saturate the Teflon chamber. The silicon substrate withthe silanol groups was put into the Teflon chamber and subjected tovapor deposition for 1 hour.

COMPARATIVE EXAMPLE 2

A silicon substrate was washed with sulfuric acid, an aqueousfluorinated hydrogen solution, and then deionized water and dried. Thesilicon substrate was put into a reactor to form a 5000 Å-thick oxidefilm thereon at a temperature of 1000-1100° C., a pressure of 1 atm, andan oxygen flow rate of 4 L/min.

Evaluation Method

PCR was carried out with PCR chips manufactured using the siliconsubstrates surface-treated in Example 1 and Comparative Examples 1 and2. The PCR chips were manufactured by forming channel structures in thesilicon substrates (lower substrates) and covering them with glasssubstrates (upper substrates) by anode bonding to form PCR chamberstherein. A heater and a sensor were attached to an outer surface of eachof the silicon substrates. An external electronic control unit wasconnected to the heater to apply voltage, and an external fan wasattached to control the temperature of the PCR chamber. An inlet forinjecting PCR reactants and an outlet for discharging PCR products wereformed in each of the upper glass substrates.

MODY3 gene was amplified through temperature cycling of denaturation,annealing, and extension in each of the PCR chips. PCR reactantscontained no additive, such as bovine serum albumin (BSA).

As a control, PCR was conducted using a polyethylene e-tube in the sameconditions as for the above PCR chips.

The PCR products from each of the PCR chips and the polyethylene e-tubewere analyzed using a Labchip (available from Agilent Co.)

The results are shown in FIGS. 2 through 5 and Table 1, wherein FIG. 2is for the silicon substrate of Example 1, FIG. 3 is for the siliconsubstrate of Comparative Example 1), FIG. 4 is for the silicon substrateof Comparative Example 2), and FIG. 5 is for the polyethylene e-tube).

TABLE 4 PCR Yield Example Surface Treatment Method (ng/μL) Example 1Treatment with two silane-containing 40.1 compounds according to thepresent invention Comparative Treatment with a kind of fluorinated 5.1Example 1 hydrocarbon Comparative Treatment with oxide 20.5 Example 2Control Polypropylene e-tube 40.2

The PCR yield for the silicon chip treated with oxide (ComparativeExample 2) is about half of the PCR yield for the polypropylene e-tube(Control). The PCR yield for the silicon chip treated with one kind offluorinated hydrocarbon that include a silane group as a linker and atrifluoromethyl group at a terminal (Comparative Example 1) is greatlylower than the PCR yield for the polypropylene e-tube. However, the PCRyield for the silicon chip surface-treated according to the presentinvention is almost the same as the PCR yield for the polypropylenee-tube.

As described above, when a surface of a silicon or glass substrate usedfor a biochemical reaction system, such as a PCR chip, is treated withthe compound of formula (1) and the compound of formula (2) according tothe present invention, non-specific binding of biomolecules to thesilicon or glass substrate is suppressed, improving the yield ofbiochemical reaction, including PCR.

In addition, according to the present invention, the composition iscoated on the substrate by vapor deposition at low temperature so thatan expensive plasma enhanced chemical vapor deposition (PECVD) systemconventionally used is not required. Furthermore, a series ofcomplicated processes, such as flowing a liquid coating composition intoa miniature 3D chip structure, washing the chip structure with acleaning solution, and drying, are not involved in the presentinvention, so that the time required for such processes is saved.

While the present invention has been particularly shown and describedwith reference to exemplary embodiments thereof, it will be understoodby those of ordinary skill in the art that various changes in form anddetails may be made therein without departing from the spirit and scopeof the present invention as defined by the following claims.

1. A biochemical reaction system, comprising: a substrate including apolymer film on a surface of the substrate, the polymer film beingformed on the surface by vapor deposition of a compound of formula (1)below and a compound of formula (2) below:(RO)₃—Si—(CH₂)_(n1)—X  (1)(RO)₃—Si—(CH₂)_(n2)—(CF₂)_(m)—X  (2) wherein R is one of a methyl groupand an ethyl group, X is one of a methyl group and a trifluoromethylgroup, n1 is an integer from 1 to 3, n2 is an integer from 1 to 10, andm is an integer from 1 to
 10. 2. The biochemical reaction system ofclaim 1 being a polymerase chain reaction (PCR) system.
 3. Thebiochemical reaction system of claim 1, wherein the compound of saidformula (1) and the compound of said formula (2) are simultaneouslydeposited by vaporization.
 4. The biochemical reaction system of claim1, wherein the compound of said formula (1) and the compound of saidformula (2) are sequentially deposited by vaporization.
 6. A compositionfor treating a surface of a substrate used in a biochemical reactionsystem, the composition comprising a compound of formula (1) below and acompound of formula (2) below:(RO)₃—Si—(CH₂)_(n1)—X  (1)(RO)₃—Si—(CH₂)_(n2)—(CF₂)_(m)—X  (2) wherein R is one of a methyl groupand an ethyl group, X is one of a methyl group and a trifluoromethylgroup, n1 is an integer from 1 to 3, n2 is an integer from 1 to 10, andm is an integer from 1 to 10.